Synthethic peptides and pharmaceutical compositions comprising them for the treatment of systemic lupus erythematosus

ABSTRACT

Synthetic peptides based on a complementarity-determining region (CDR) of the heavy or light chain of a pathogenic anti-DNA monoclonal antibody that induces a systemic lupus erythematosus (SLE)-like disease in mice, and analogs, and salts and chemical derivatives thereof; dual peptides comprising two such peptides or analogs covalently linked to one another either directly or through a short linking chain; peptide polymers comprising a plurality of sequences of said peptide or analog thereof; and peptide polymers attached to a macromolecular carrier, are disclosed, and pharmaceutical compositions comprising them for the treatment of SLE in humans.

FIELD OF THE INVENTION

[0001] The present invention relates to synthetic peptides and topharmaceutical compositions comprising them useful for the treatment ofsystemic lupus erythematosus (SLE) in humans.

BACKGROUND OF THE INVENTION

[0002] Autoimmune diseases are characterized by immune responses thatare directed against self antigens. These responses are maintained bythe persistent activation of self-reactive T lymphocytes. T lymphocytesare specifically activated upon recognition of foreign and/or selfantigens as a complex with self major histocompatibility complex (MHC)gene products on the surface of antigen-presenting cells (APC).

[0003] Systemic lupus erythematosus (SLE) is an autoimmune disease ofunknown origin and cure. Despite the extensive research on themechanisms underlying the induction and development of SLE, theinformation available on the etiology of the disease is very limited dueto the heterogeneity of SLE patients on one hand, and the lack of anexperimental model in which the induction of the disease could becontrolled, on the other hand.

[0004] The cause of SLE is unknown and it has heterogeneous clinicalmanifestations. Furthermore, no specific treatment aimed towards theprevention or cure of SLE is available. Despite the extensive researchon the mechanisms underlying the induction of SLE, the information onthe etiology of the disease is very limited. Studies on SLE have beenperformed until recently using peripheral blood lymphocytes (PBL) ofpatients at different clinical stages and under various treatmentprotocols. Alternatively, murine strains that develop spontaneousSLE-like disease were investigated as a model for SLE. This kind ofanalysis led to incomplete and confusing interpretations of the role ofvarious immunological and non-immunological factors in either inducingor sustaining the disease, mainly due to the heterogeneity of patientson one hand and the inability to control the induction phase of thedisease in murine SLE strains on the other hand.

[0005] Several years ago, an animal model of SLE has been established inthe laboratory of one of the present inventors. This model, based on theconcept of the idiotypic network, developed a wide spectrum oflupus-related autoantibodies and clinical manifestations (Mendlovic etal., 1988). The induction was carried out by the immunization of mousestrains that do not develop any spontaneous autoimmune disorders, with ahuman anti-DNA monoclonal antibody (mAb) which bears a common idiotypetermed 16/6 Id (Shoenfeld et al., 1983). Following immunization, themice produced antibodies specific to the 16/6 Id, antibodies that bearthe 16/6 Id and antibodies directed against different nuclear antigens(dsDNA, ssDNA, Sm, ribonucleoprotein (RNP), Ro, La and others). Theserological findings were associated with leukopenia, elevatederythrocyte sedimentation rate, proteinuria, abundance of immunecomplexes in the kidneys and sclerosis of the glomeruli (Mendlovic etal., 1988), which are typical manifestations of SLE. The presentinventors have further shown that the experimental disease could beinduced by a murine anti-16/6 Id mAb (Mendlovic et al., 1989) and by themouse anti-anti 16/6 Id (16/6Id+) mAb (Waisman et al., 1993). Theinduction of the disease is genetically controlled, and thus is straindependent (Mendlovic et al., 1990). This unique model for the inductionof experimental SLE provides the appropriate tools to clearly dissectthe different steps and the linked immune parameters involved in theinduction and development of SLE.

[0006] SLE is a systemic autoimmune disease characterized by theformation of autoantibodies against self-antigens, such as DNA, Sm, Ro,La, RNP, cardiolipin and histones. The etiology of SLE is unknown, andunderstanding the mechanism by which these self-antibodies arise mightprovide insight to this problem. For this purpose, the present inventorshave produced a variety of monoclonal autoantibodies derived from C3H.SWmice in which experimental SLE was induced. As a rule, the monoclonalautoantibodies that were capable of eliciting antibodies that bear the16/6 Id or react with it were found to be pathogenic and thus capable ofinducing experimental SLE (Fricke et al., 1990; Sthoeger et al., 1993).Later on, the variable (V) regions of nine autoantibodies that bindeither DNA or HeLa nuclear extract (NE), isolated from the C3H.SW micewith experimental SLE, were sequenced (Waisman and Mozes, 1993).Monoclonal antibodies with different specificity were analyzed in anattempt to determine the connections between the differentautoantibodies. Three mAb were found to bind DNA, and were shown toexhibit sequence characteristics of pathogenic anti-DNA antibodies. Oneof these mAb, designated 2C4C2, was shown to use a heavy (H) chain Vregion gene (V_(H)) identical to the V_(H) of anti-DNA mAb isolated fromother lupus-prone mice, namely (NZB x NZW)F₁. The light (L) chain Vregion gene (V_(L)) of mAb 2C4C2 is 98% homologous to the V_(L) ofanother anti-DNA mAb, also isolated from (NZB x NZW)F₁ mice. The othertwo anti-DNA mAb, designated 5G12-4 and 5G12-6, share 93% of their V_(H)sequences with that of mAb 2C4C2. Six mAb bound proteins of HeLa NE. Thenine mAb use a total of five V_(H) and four V_(L) germ-line genes,demonstrating that the autoantibodies induced in mice with experimentalSLE do not originate from one B cell clone. Three of the nine V_(H) andV_(L) were identical in sequence to germ-line genes, while at leastthree others had somatic mutations. The latter suggests that theseautoantibodies arise in mice by both usage of existing (pre-immune) Bcells, and through an antigen-driven process. Furthermore, it appearsthat autoantibodies found in mice with experimental SLE use geneticelements similar to those used by mAb that were isolated from mousestrains which develop lupus spontaneously.

[0007] T cells play an important role in the induction and developmentof experimental SLE. Thus, T cell lines and clones specific to the 16/6Id were shown to induce experimental SLE in syngeneic recipientssimilarly to the 16/6 antibody. Therefore, following the inoculation ofthe activated cells of the lines, the mice developed both the serologyand the renal damage which is typical to SLE (Fricke et al., 1991).Furthermore, a 16/6 Id specific T cell line of C3H.SW origin induced SLEin C57BL/6 mice that were shown to be resistant to the induction of thedisease following injections with either the 16/6 Id or the anti-16/6 IdmAb (Mendlovic et al., 1990).

[0008] In an attempt to identify the pathogenic region of the 16/6 Id,(Fab′)₂ fragments were prepared of the 16/6 Id mAb and were found toretain the specificity and pathogenic capacity of the whole 16/6 Idmolecule (Ruiz et al., 1994).

[0009] The mAb 5G12 that was isolated from mice with experimental SLEand was shown to bind DNA and bear the 16/6 Id, is capable of inducingexperimental SLE in mice (Waisman et al., 1993). T cells that reactspecifically to mAb by proliferation, are probably reacting to peptidesrepresenting sequences from their complementarity-determining regions(CDR). It is very likely that the T cells recognize the V regions of theabove antibodies since they do not react with other antibodies thatcarry the same constant region but have different specificities. Withinthe variable region, the regions with the highest probability to berecognized are the CDR, since those are the regions that differ the mostbetween the various antibodies. The CDR regions of the V_(H) sequencesof the nine pathogenic murine mAb mentioned above that induce SLE inmice, are boxed in FIG. 1 of Waisman and Mozes, 1993, in which thecomplete nucleotide and deduced amino acid sequences for the V_(H) ofthe nine mAb are presented.

SUMMARY OF THE INVENTION

[0010] It is an object of the present invention to provide means forspecific treatment of patients with SLE.

[0011] For this purpose, the invention provides peptides and analogsthereof based on the CDR regions of pathogenic monoclonal autoantibodiesisolated from mice with experimental SLE.

[0012] Thus, in one aspect, the invention relates to a synthetic peptideselected from the group consisting of:

[0013] (i) a peptide of at least 12 and at most 30 amino acid residuesbased on a complementarity-determining region (CDR) of the heavy orlight chain of a pathogenic anti-DNA monoclonal antibody that induces asystemic lupus erythematosus (SLE)-like disease in mice (hereinafterCDR-based peptide), a salt or a chemical derivative thereof;

[0014] (ii) an analog of a CDR-based peptide defined in (i), a salt or achemical derivative thereof;

[0015] (iii) a dual synthetic peptide comprising two such peptides of(i) or analogs of (ii) covalently linked to one another either directlyor through a short linking chain;

[0016] (iv) a peptide polymer comprising a plurality of sequences ofsaid peptide (i) or analog thereof (ii); and

[0017] (v) a peptide polymer (iv) attached to a macromolecular carrier.

[0018] In one embodiment of this aspect, the synthetic peptide iscapable of:

[0019] (i) inhibiting specifically the proliferative response andcytokine secretion of T lymphocytes of mice that are high responders toSLE-inducing autoantibodies; or

[0020] (ii) inhibiting development of SLE in mice that are susceptibleto SLE-induction by pathogenic autoantibodies.

[0021] The synthetic peptides and analogs thereof according to theinvention may be selected from the group consisting of peptides havingthe sequences I to V herein, wherein:

[0022] (i) the peptide of sequence I has the formula:TGYYX₁X₂X₃X₄X₅QSPEKSLEWIG [I]

[0023] wherein X₁ is Met, Ala or Val; X₂ is Gin, Asp, Glu or Arg; X₃ isTrp or Ala; X₄ is Val or Ser; and X₅ is Lys, Glu or Ala;

[0024] (ii) the peptide of sequence II has the formula:EINPSTGGX₆X₇X₈X₉X₁₀X₁₁X₁₂KAKAT [II]

[0025] wherein X₆ and X₇ are each Thr, Val or Ala; X₈ is Tyr or Phe; X₉is Asn or Asp; X₁₀ is Gin or Glu; X₁₁ is Lys or Glu, and X₁₂ is Phe orTyr;

[0026] (iii) the peptide of sequence III has the formula:YYCARX₁₃X₁₄X₁₅X₁₆PYAX₁₇X₁₈YWGQGS [III]

[0027] wherein X₁₃ is Phe, Thr or Gly; X₁₄ is Leu, Ala or Ser; X₁₅ isTrp or Ala; X₁₆ is Glu or Lys; X₁₇ is Met or Ala, and X₁₈ is Asp, Lys orSer;

[0028] (iv) the peptide of sequence IV has the formula:GYNX₁₉X₂₀X₂₁X₂₂X₂₃X₂₄SHGX₂₅X₂₆LEWIG [IV]

[0029] wherein X₁₉ is Met or Ala; X₂₀ is Asn, Asp or Arg; X₂₁ is Trp orAla; X₂₂ is Val or Ser; X₂₃ is Lys or Glu; X₂₄ is Gln or Ala; X₂₅ is Lysor Glu, and X₂₆ is Ser or Ala; and

[0030] (v) the peptide of sequence V has the formula:YYCARX₂₇X₂₈X₂₉YGX₃₀X₃₁X₃₂GQGTL [V]

[0031] wherein X₂₇ is Ser or Phe; X₂₈ is Gly or Ala; X₂₉ is Arg, Ala orGlu; X₃₀ is Asn or Asp; X₃₁ is Tyr or Phe, and X₃₂ is Trp, His or Ala.

[0032] In preferred embodiments, peptides I to V have the sequencesIa-Va herein: TGYYMQWVKQSPEKSLEWIG (Ia) EINPSTGGTTYNQKFKAKAT (IIa)YYCARFLWEPYAMDYWGQGS (IIIa) GYNMNWVKQSHGKSLEWIG (IVa) YYCARSGRYGNYWGQTL(Va)

[0033] Peptides Ia to IIIa are based on the CDR1, CDR2 and CDR3 regions,respectively, of the V_(H) chain of mAb 5G12, and peptides IVa and Vaare based on the CDR1 and CDR3 regions, respectively, of the V_(H) chainof mAb 2C4C2 (Waisman and Mozes, 1993).

[0034] In another aspect, the invention relates to pharmaceuticalcompositions for the treatment of SLE comprising a synthetic peptide orpeptide polymer of the invention and a pharmaceutically acceptablecarrier.

[0035] In still another aspect, the invention relates to a method oftreatment of a SLE patient comprising administering to a SLE patient aneffective amount of a synthetic peptide or peptide polymer of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIGS. 1A-B show the presence of anti-DNA antibodies in sera ofSJL (1A) and BALB/c (1B) mice immunized with mAb 5G12, peptides Ia andIIIa and a control peptide 278, or non-immunized. Sera of individual SJLor BALB/c mice immunized with either one of the indicated antigens,taken three months after the booster injection, and sera of age-matchednaive mice, were tested for anti-ssDNA antibody titers. Followingincubation with the diluted sera, goat anti-mouse IgG (γ-chain specific)conjugated to peroxidase was added. Results were expressed as mean OD ofeach mouse group.

[0037] FIGS. 2A-B show the presence of HeLa anti-nuclear extract (NE)antibodies in the sera of SJL (2A) and BALB/c (2B) mice immunized withsame antigens as in FIG. 1.

[0038] FIGS. 3A-B show the presence of anti-RNP, Sm, Ro and Laantibodies in the sera of SJL (3A) and BALB/c (3B) mice immunized withpeptides Ia and IIIa or with control peptide 278, and normal mice.

[0039] FIGS. 4A-B show the presence of anti-DNA (4A) and anti-HeLa NE(4B) antibodies in the sera of BALB/c mice tolerized with peptide Ia orwith control peptide p307, and immunized with either peptide Ia or mAb5G12.

[0040]FIGS. 5a-b show in vivo inhibition of lymph node cell (LNC)proliferation responses in BALB/c (5 a) and SJL (5 b) mice to theCDR-based peptides Ia and IIIa, respectively, following treatment withthe latter.

[0041]FIGS. 6a-b show in vivo inhibition of LNC to mAb 5G12 in BALB/c (6a) or SJL (6 b) mice treated with peptide Ia and IIIa, respectively.

[0042]FIGS. 7a-b show in vivo inhibition of LNC proliferation to thehuman monoclonal anti-DNA 16/6 Id antibody in BALB/c (7 a) and SJL (7 b)mice treated with peptide Ia and IIIa, respectively.

[0043]FIG. 8 shows binding of peptides Ia and IIIa to the surface ofsplenic antigen-presenting cells of different mouse strains.

[0044]FIG. 9 shows antibody titers in sera of SLE patients and healthyhuman controls by testing their sera for the ability to bind thepeptides Ia, IIa and IIIa, or mAb 5G12 or a control peptide.

DETAILED DESCRIPTION OF THE INVENTION

[0045] The present invention relates to synthetic peptides that arebased on the CDR of monoclonal pathogenic autoantibodies isolated frommice with experimental SLE. Such monoclonal antibodies are obtained fromsupernatants of hybridomas produced by fusion, for example, of spleencells of C3H.SW mice immunized with an anti-16/6 Id mAb, with X63.653plasmacytoma cells (Waisman and Mozes, 1993).

[0046] Examples of such peptides are those of formulas Ia to Va herein,based on, respectively, the CDR1, CDR2 and CDR3 regions of the heavychain of mAb 5G12 and the CDR1 and CDR3 regions of the heavy chain ofmAb 2C4C2 (Waisman and Mozes, 1993), and analogs thereof.

[0047] Analogs of parent peptides Ia-Va contemplated by the inventioninclude substitution, deletion and addition analogs as described herein.Substitution analogs have amino acid substitutions at differentpositions, these substitutions being made based on the volume,hydrophobic-hydrophilic pattern and charge of the amino acids.

[0048] Amino acids may be divided along the lines of volume,hydrophobic-hydrophilic pattern and charge. With respect to volume,those of ordinary skill in the art understand that the amino acids withthe largest volume are Trp, Tyr, Phe, Arg, Lys, Ile, Leu , Met and His,while those with the smallest volumes are Gly, Ala, Ser, Asp, Thr andPro, with others being in between.

[0049] With respect to hydrophobic-hydrophilic pattern, it is well knownthat the amino acids Gly, Ala, Phe, Val, Leu, Ile, Pro, Met and Trp arehydrophobic, whereas all of the remaining amino acids are hydrophilic.Among the hydrophilic amino acids, Ser, Thr, Gln, and Tyr have nocharge, while Arg, Lys, His and Asn have a positive charge and Asp andGlu have negative charges.

[0050] In selecting peptides to be tested for their potential ininhibiting the proliferative response of T lymphocytes of mice that arehigh responders to SLE-inducing autoantibodies, it is important that thesubstitutions be selected from those which cumulatively do notsubstantially change the volume, hydrophobic-hydrophilic pattern andcharge of the corresponding portion of the unsubstituted parent peptide.Thus, a hydrophobic residue may be substituted with a hydrophilicresidue, or vice-versa, as long as the total effect does notsubstantially change the volume, hydrophobic-hydrophilic pattern andcharge of the corresponding unsubstituted parent peptide.

[0051] It should be understood that other modifications of the peptidesand analogs thereof are also contemplated by the present invention.Thus, the peptide or analog of the present invention is intended toinclude a “chemical derivative” thereof which retains at least a portionof the function of the peptide which permits its utility in preventingor inhibiting T cell proliferative responses and autoimmune disease.

[0052] A “chemical derivative” of a peptide or analog of the presentinvention contains additional chemical moieties not normally a part ofthe peptide. Covalent modifications of the peptide are included withinthe scope of this invention. Such modifications may be introduced intothe molecule by reacting targeted amino acid residues of the peptidewith an organic derivatizing agent that is capable of reacting withselected side chains or terminal residues. Many such chemicalderivatives and methods for making them are well known in the art.

[0053] Also included in the scope of the invention are salts of thepeptides and analogs of the invention. As used herein, the term “salts”refers to both salts of carboxyl groups and to acid addition salts ofamino groups of the peptide molecule. Salts of a carboxyl group may beformed by means known in the art and include inorganic salts, forexample, sodium, calcium, ammonium, ferric or zinc salts, and the like,and salts with organic bases such as those formed for example, withamines, such as triethanolamine, arginine, or lysine, piperidine,procaine, and the like. Acid addition salts include, for example, saltswith mineral acids such as, for example, hydrochloric acid or sulfuricacid, and salts with organic acids, such as, for example, acetic acid oroxalic acid. Such chemical derivatives and salts are preferably used tomodify the pharmaceutical properties of the peptide insofar asstability, solubility, etc., are concerned.

[0054] Examples of peptides and analogs thereof are as follows:

[0055] (i) Peptide Ia of the formula:1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20T G Y Y M Q W V K Q  S  P  E  K  S  L  E  W  I  G (Ia)

[0056] and substitution analogs thereof in which Met at position 5 issubstituted by either Ala or Val; Gln at position 6 is substituted byeither Asp, Glu or Arg; Trp at position 7 is substituted by Ala; Val atposition 8 by Ser; and Lys at position 9 is substituted by either Glu orAla; and deletion analogs thereof in which up to 5 amino acid residuesare deleted from the C-terminal of peptide Ia.

[0057] (ii) Peptide IIa of the formula:1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20E I N P S T G G T T  Y  N  Q  K  F  K  A  K  A  T (IIa)

[0058] and substitution analogs thereof in which Thr in positions 9 and10 are each substituted by either Val or Ala; Tyr at position 11 issubstituted by Phe; Asn at position 12 is substituted by Asp; Gln atposition 13 by Glu; Lys at position 14 by Glu; and Phe at position 15 byTyr, and deletion analogs thereof in which up to 5 amino acid residuesare deleted from the C-terminal of peptide IIa

[0059] (iii) Peptide IIIa of the formula:1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Y Y C A R F L W E P  Y  A  M  D  Y  W  G  Q  G  S (IIIa)

[0060] and substitution analogs thereof in which Phe at position 6 issubstituted by either Thr or Gly; Leu at position 7 is substituted byeither Ala or Ser; Trp at position 8 is substituted by Ala; Glu atposition 9 is substituted by Lys; Met at position 13 by Ala; and Asp atposition 14 by either Lys or Ser; and deletion analogs thereof in whichup to 5 amino acid residues are deleted from the C-terminal of peptideIIIa.

[0061] (iv) Peptide IVa of the formula:1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19G Y N M N W V K Q S  H  G  K  S  L  E  W  I  C (IVa)

[0062] and substitution analogs thereof in which Met at position 4 issubstituted by Ala; Asn at position 5 is substituted by either Asp orArg; Trp at position 6 is substituted by Ala; Val at position 7 by Ser;Lys at position 8 by Glu; Gln at position 9 by Ala; Lys at position 13by Glu; and Ser at position 14 by Ala; and deletion analogs thereof inwhich up to 5 amino acid residues are deleted from the C-terminal ofpeptide IVa.

[0063] (v) Peptide Va of the formula: 1 2 3 4 5 6 7 8 9 10 11 12 13 1415 16 17 18 Y Y C A R S G R Y G  N  Y  W  G  Q  G  T  L (V)

[0064] and substitution analogs thereof in which Ser at position 6 issubstituted by Phe; Gly at position 7 is substituted by Ala; Arg atposition 8 is substituted by either Ala or Glu; Asn at position 1 issubstituted by Asp; Tyr at position 12 by Phe; and Trp at position 13 byeither His or Ala; and deletion analogs thereof in which up to 5 aminoacid residues are deleted from the C-terminal of peptide Va.

[0065] Once an analog in accordance with the present invention isproduced, its ability to inhibit the proliferative response of Tlymphocytes of mice that are high responders to SLE-inducingautoantibodies may be readily determined by those of ordinary skill inthe art without undue experimentation using tests such as thosedescribed herein. One test which may be readily conducted is for theability of substituted peptides to inhibit in vitro the proliferativeresponses of certain T cell lines and clones specific to SLE-inducingautoantibodies. The T cell lines and clones may, for example, be the Tcell lines and clones specific to the 16/6 Id mAb (Fricke et al., 1991)established from immunized lymph node cells of mice by previouslydescribed methodology (Axelrod and Mozes, 1986). Cells are exposed tothe stimulating antibody presented on irradiated syngeneic spleen cellsin the presence of enriched medium every two weeks. The T cell lines arecloned by the standard limiting dilution technique.The proliferativeresponses of these T cell lines and clones are tested, for example, bythe method described in Materials and Methods, section (g), herein.

[0066] Another test which can be conducted in order to select analogshaving the desired activity is to test for the ability of thesubstituted peptides to inhibit the ability of the T cell lines andclones to provide help to peptide-specific B cells in the presence ofthe parent peptide. The substituted peptides may also be tested fortheir ability to bind directly, following biotinylation, to MHC Class IIproducts on antigen-presenting cells of the relevant strains. For thispurpose, N-terminal biotinylation of the relevant peptides is performedat 0° C. with an excess of biotin-N-hydroxysuccinimide in aqueoussolution (Mozes et al., 1989). Mouse splenic adherent cells or humanperipheral blood lymphocyte (PBL)-adherent cells (1×10⁶/sample) areincubated with biotinylated peptides in PBS containing 0.1% bovine serumalbumin (PBS/BSA) at 37° C. for 20 hr, followed by incubation withphycoerythrin-streptavidin for 30 min at 4° C. After each incubation,the cells are washed twice with the above solution. Thereafter, thecells are analyzed by flow cytometry using FACScan. In each analysis, aminimum of 5000 cells are examined (for above procedures, see, forexample, Mozes et al., 1989; Zisman et al., 1991).

[0067] A further test which can be conducted is to test for the abilityof the analogs to inhibit cytokine secretion by the T cell line or by Tlymphocytes oh lymph nodes of mice that are high responders toSLE-inducing autoantibodies. The cytokines are detected as follows: IL-1activity is assessed either by ELISA using a pair of capture anddetecting antibodies (as described below for IL-4, IL-6, IL-10) or usingthe LBRM-33(1A5) assay (Conlon, 1983) in which 1A5 cells are stimulatedin the presence of phytohemagglutinin (PHA), with either supernatants orrecombinant IL-1 at various concentrations to secrete IL-2. Following anovernight incubation, supernatants of 1A5 cells are transferred to theIL-2 dependent cytotoxic T lymphocyte (CTLL) line. Stimulation of theCTLL line by IL-2 is measured after 24 hr by incorporation of³[H]-thymidine. IL-2 is directly detected using the IL-2 dependent CTLLline or by ELISA. Levels of IL-4, IL-6, IL-10, INFγ and TNFα in thesupernatants are determined by ELISA using antibodies to the variouscytokines (Phamingen, San Diego, Calif., USA) according to themanufacturers instructions.

[0068] Peptides which test positive in one or more of these in vitrotests will provide a reasonable expectation of in vivo activity.However, in vivo tests can also be conducted without undueexperimentation. Thus, for example, adult mice may be injected with thecandidate peptide at either day −3 or day 0. The mice are then immunizedwith the disease-inducing autoantibody or with the peptide. Ten dayslater, lymph node cells of the mice are tested for their ability toproliferate to the immunogen in order to find out the inhibitorycapacity of the candidate peptide.

[0069] Another such in vivo animal test consists in measuring thetherapeutic activity directly in the murine model in vivo for theproduction of SLE as described above. The peptides can be injected intothe mice in which experimental SLE is induced by different routes atdifferent dosages and at different time schedules. In order to determinethe pharmnacokinetic parameters of the analogs, including volume ofdistribution, uptake into antigen-presenting cells and clearance, onecan use biotinylated derivatives of the analogs. The concentration ofthe soluble fraction of the analogs in the various body fluids can bedetermined by ELISA, using avidin-coated plates and specificanti-peptide antibodies. Cell bound analogs can be analyzed by FACS,using fluorochromo-conjugated avidin or streptavidin. Furthermore, thetreated mice can be tested periodically in order to determine the effectof the peptides on the autoantibody responses and on diseasemanifestations elicited in the mice by the SLE-inducing autoantibody.

[0070] Another in vivo procedure consists in tolerizing newborn micewith the candidate peptide followed by immunization of the mice with thepathogenic autoantibody, such as 16/6 Id+, or with the same peptide, andfollowing the disease manifestations, such as serological findingsassociated with leukopenia, elevated erythrocyte sedimentation rate,proteinuria, abundance of immune complexes in the kidneys and sclerosisof the glomeruli.

[0071] It can thus be seen that, besides the preferred embodiments whichhave been shown to be operable in the examples herein, those of ordinaryskill in the art will be able to determine additional analogs which willalso be operable following the guidelines presented herein without undueexperimentation.

[0072] A relatively simple in vitro test can also be conducted in orderto assay for the expected therapeutic efficacy of any given substitutedpeptide on any given SLE patient. In order to assess the ultimate goalof producing peptides that will bind with high affinity to theappropriate MHC Class II molecules but will not lead to furtheractivation of T cells and will therefore have a therapeutic effect onSLE patients, the peptides may be assayed, following biotinylation, fortheir ability to bind directly to HLA Class II products onantigen-presenting cells in the peripheral blood lymphocytes of the SLEpatients. Healthy control donors and control peptides may be used insuch assays to verify their specificity.

[0073] A preferred form of the therapeutic agent of the invention is apeptide selected from the group of peptides of formulas I to V herein,including peptides Ia to Va and substitution and/or deletion analogsthereof.

[0074] Another preferred form of the therapeutic agent in accordancewith the present invention is the form of a multi-epitope singlepeptide. Thus, in a preferred embodiment, dual petides consisting of twodifferent peptides selected from the group of peptides of formula 1-Vherein, are covalently linked to one another, such as by a short stretchof alanine residues or by a putative site for proteolysis by cathepsin.See, for example, U.S. Pat. No. 5,126,249 and European Patent 495,049with respect to such sites. This will induce site-specific proteolysisof the preferred form into the two desired analogs. Alternatively, anumber of the same or different peptides of the present invention may beformed into a peptide polymer, such as, for example, polymerization ofthe peptides with a suitable polymerization agent, such as 0.1%glutaraldehyde (Audibert et al. (1981), Nature 289:593). The polymerwill preferably contain from 5 to 20 peptide residues. Such peptidepolymers may also be formed by crosslinking the peptides or attachingmultiple peptides to macromolecular carriers. Suitable macromolecularcarriers are, for example, proteins, such as tetanus toxoid, and linearor branched copolymers of amino acids, such as a linear copolymer ofL-alanine, L-glutamic acid and L-lysine and a branched copolymer ofL-tyrosine, L-glutamic acid, L-alanine and L-lysine (T,G)-A-L-, ormultichain poly-DL-alanine (M. Sela et al. 1955, J. Am. Chem. Soc.77:6175). The conjugates are obtained, for example, by first couplingthe peptide with a water-soluble carbodiimide, such as1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride, and thenperforming the conjugation with the macromolecular carrier as describedby Muller, G. M. et al. (1982) Proc. Natl. Acad. Sci. USA 79:569. Thecontents of the coupled peptide in each conjugate are determined byamino acid analysis, in comparison to the composition of the carrieralone.

[0075] According to one embodiment of the present invention, one or moreactive peptides may be attached to a suitable macromolecular carrier ormay be polymerized in the presence of glutaraldehyde.

[0076] The peptides, polymers thereof or their conjugates with suitablemacromolecular carriers, will be given to patients in a form thatinsures their bioavailability, making them suitable for treatment. Ifmore than one peptide analog is found to have significant inhibitoryactivity, these analogs will be given to patients in a formulationcontaining a mixture of the peptides.

[0077] The invention further includes pharmaceutical compositionscomprising at least one synthetic peptide according to the invention, aconjugate thereof with a suitable macromolecular carrier or a polymerthereof optionally with a pharmaceutically acceptable carrier.

[0078] Any suitable route of administration is encompassed by theinvention, including oral, intravenous, subcutaneous, intraarticular,intramuscular, inhalation, intranasal, intrathecal, intraperitoneal,intradermal, transdermal or other known routes, including the enteralroute.

[0079] The dose ranges for the administration of the compositions of thepresent invention should be large enough to produce the desired effect,whereby, for example, an immune response to the SLE-inducingautoantibody, as measured by T cell proliferation in vitro, issubstantially prevented or inhibited, and further, where the disease issignificantly treated. The doses should not be so large as to causeadverse side effects, such as unwanted cross reactions, generalizedimmunosuppression, anaphylactic reactions and the like.

[0080] Effective doses of the peptides of this invention for use intreating SLE are in the range of about 1 μg to 100 mg/kg body weight.The dosage administered will be dependent upon the age, sex, health, andweight of the recipient, kind of concurrent treatment, if any, frequencyof treatment, and the nature of the effect desired.

[0081] The synthetic peptides and analogs of the invention, particularlythose of sequences I to V herein, are aimed at inhibiting or suppressingspecific antigen responses of SLE patients, without affecting all otherimmune responses. This approach is of the utmost importance since mostdiagnosed patients are young women that have to be treated for manyyears and the currently accepted treatment for SLE involvesadministration of immuno-suppressive agents, such as corticosteroidsand/or cytotoxic drugs, that are both non-specific and have multipleadverse side effects.

[0082] The present invention will now be described in more detail in thefollowing non-limiting Examples and the accompanying figures:

EXAMPLES Materials and Methods

[0083] a) Mice: Mice (BALB/c and SJL/J) were obtained from the JacksonLaboratory, Bar Harbor, Me., USA and from Olac, Show's farm, BicesperOxon, England. Mice were used at the age of 6-12 weeks. In some studiesneonatal mice were also used.

[0084] b) Human mAb 16/6 Id: The human mAb 16/6 is an anti-DNA antibodyoriginally of the IgM isotype and switched in culture to IgGl. The mAbwas derived from a patient and expresses a common idiotype, the 16/6 Id(Shoenfeld et al., 1983; Mendlovic et al., 1988). The hybridoma cellssecreting this mAb are routinely grown in culture, and the antibody isisolated from culture supernatants using an affinity column of Protein Gcoupled to Sepharose™.

[0085] c) Production of mouse mAb 5G12 and 2C4C2: Experimental SLE wasinduced in C3H.SW female mice by immunization with the previouslydescribed murine anti-16/6 Id mAb (Mendlovic et al., 1989). Four monthslater, two mice were sacrificed and their spleen cells were fused withX63.653 plasmacytoma cells. Hybridoma cells that secreted autoantibodieswere cloned by limiting dilution in 96-well microtiter plates. Thesequence characteristics of nine monoclonal autoantibodies secreted bynine of the hybridoma clones were characterized (Waisman and Mozes,1993). The mAb designated 5G12 and 2C4C2 were isolated and affinitypurified from the hybridoma supernatants using a goat anti-mouseIg-Sepharose™ 4B column. The 5G12 mAb was found to be an anti-DNA mAbthat bear the 16/6 Id and have the IgG2a isotype. The 2C4C2 mAb wasfound to be an anti-DNA and anti-cardiolipin mAb and to be of the IgMisotype. The nucleotide and deduced amino acid sequences for the V_(H)of both 5G12 and2C4C2 mAb are presented in FIG. 1 of Waisman and Mozes,1993, in which figure the CDR regions are boxed.

[0086] d) Induction of experimental SLE in mice: Mice were injected withthe human monoclonal 16/6 Id (1 μg/mouse) or the murine 16/6 Id mAb,e.g. mAb 5G12 (20 μg/mouse), in complete Freund's adjuvant in the hindfootpads. Three weeks following injection, the mice were boosted withthe same amount of the immunizing antibody in phosphate-buffered saline(PBS).The mice were then tested for autoantibody production and clinicalmanifestations characteristic of experimental SLE.

[0087] e) Detection of SLE-associated clinical manifestations: Theerythrocyte sedimentation rate was determined by diluting theheparinized blood in PBS at a ratio of 1:1. The diluted blood was thenpassed to a microsampling pipette and the sedimentation was measured 6hours later. White blood cell counts were determined after the hemolysisof heparinized blood. Proteinuria was measured in a semi-quantitativemanner, using a Combistix kit (Ames, Stoke Poges, Slough, U.K.).Immunohistology was performed by incubation of fixed frozen cryostatsections with FITC-labeled antibodies to mouse Ig. Staining wasvisualized via use of a fluorescent microscope.

[0088] f) Enzyme-linked immunosorbent assay (ELISA): ELISA was utilizedfor the detection and quantitation of antibodies in experimental mice,and in humans. Polystyrene microtiter plates were coated with therelevant antigen or antibody, and sera dilutions or supernatants derivedfrom the human or mouse cell cultures were added to the blocked plates.Specific binding was determined following the addition ofperoxidase-conjugated antibodies against the appropriate immunoglobulin(Ig) (e.g. goat anti-human or goat anti-mouse peroxidase-conjugatedantibodies) and the peroxidase substrate. Optical densities were read at414 nm using an ELISA reader.

[0089] g) Proliferative responses of splenic and lymph node cells: Cells(0.5×10⁶/well) derived from the spleen and lymph nodes of treated anduntreated mice were cultured in microtiter plates in the presence ofdifferent concentrations of the various immunizing pathogenicautoantibodies. At the end of 96 hours incubation, 0.5 μCi of³H-thymidine was added for an additional 18 hours, after which cellswere harvested and radioactivity was counted.

[0090] h) Treatment of experimental mice: In order to either preventinduction of experimental SLE or to cure mice afflicted with thedisease, the following procedures were used: (i) Newborn mice weretolerized with a peptide of the invention (100 μg of the peptide in PBS,intraperitoneally at 24 and 72 hours after birth). Six weeks later, themice were immunized with the pathogenic autoantibody, e.g. 5G12 (16/6Id+) and examined for disease manifestations; (ii) A first group ofadult mice was injected with various concentrations of the peptidesbefore disease induction with the pathogenic autoantibody or pathogenicT cell line; another group was injected with the peptides to be testedfor their therapeutic effect six weeks following immunization at thepeak of the serological response; and a further group was treated at 4-6months post-immunization after the establishment of the overt SLEdisease. The number of injections with the peptides was determined basedon their effect on the disease induction and progression. The effect ofthe peptide treatment on T cell proliferation, on the autoantibodyproduction and on the disease manifestations was then evaluated.

[0091] i) Proliferative responses of T cell lines and clones: T celllines and clones specific to the 16/6 Id were established from immunizedlymph node cells as previously described (Axelrod and Mozes, 1986).Cells were exposed to the stimulating antibody presented on irradiatedsyngeneic spleen cells in the presence of enriched medium every twoweeks. The T cell lines were cloned by the limiting dilution technique.Cells (10⁴/well) were cultured with 0.5×10⁶ irradiated (3000 rad)syngeneic spleen cells in the presence of different concentrations ofeither the specific stimulator of the line or control reagents. At theend of 48 hours incubation, 0.5 μCi of ³H-thymidine were added for anadditional 18 hours, after which cells were harvested and radioactivitywas counted.

[0092] j) Proliferation and cytokine production by peripheral bloodlymphocytes (PBL): PBL from human SLE patients and of the appropriatecontrol donors (2×10⁵/well) were cultured in microtiter plates inenriched medium containing 10% pooled AB sera in the presence of thehuman or mouse monoclonal 16/6Id antibody, in the presence of peptidesof the invention or in the presence of phytohemagglutinin (PHA). Therate of proliferation was evaluated by the incorporation of³[H]-thymidine in the cell culture. Non-relevant peptides were used asspecificity controls. Antigen and mitogen-stimulated cytokine productionwas quantitated in the supernatants of the above cultures using eitherthe cytokine-dependent lines or the appropriate pairs of antibodies inELISA assays. Inhibition of the proliferative responses was performediii vitro by adding increasing doses of the tested peptide analogs intothe proliferative culture mixtures.

[0093] k) Human T cell lines and clones: Human T cell lines specific tothe 16/6Id may be established from PBL of either SLE patients orcontrols following stimulation in vitro with either the human or mousemAb 16/6 Id or the peptides. The maintenance and cloning of the lineswas performed similarly to that described above for the murine T celllines, with the exception that the stimulation was performed usingeither autologous irradiated cells or EBV-transformed lines ofautologous PBL (used as antigen-presenting cells).

[0094] l) Biotinylation of peptides: N-terminal biotinylation of thepeptides was performed in 0.1N sodium bicarbonate solution at roomtemperature, with excess of biotinamnidocaproate N-hydroxysuccinimideester (Sigma, St. Louis, Mo.) dissolved in 1-methyl-2-pyrrolidone(Sigma).

[0095] m) Direct binding of biotinylated peptides to APC: Spleen cellssuspended in RPMI 1640 medium containing 10% FCS were incubated in Petridishes for 60 min at 37° C. Thereafter, non-adherent cells were removed,the plates were washed, and the adherent cells were collected from theplates using a rubber policeman (Costar, Mass., USA). These cells(1×10⁶/100 μl/tube) were incubated with the biotinylated peptides in PBScontaining 0.1% BSA (high purity grade, Amresco, Ohio, USA) for 16 hr at37° C., followed by incubation with phycoerythrin (PE)-streptavidin(Jackson ImmunoResearch) for 30 min at 4° C. Thereafter the samples wereincubated with biotinylated anti-streptavidin (1:60, VectorLaboratories, Burlingame, Calif.) and for an additional period withPE-streptavidin, all for 30 min at 4° C. The cells were washed twicewith cold PBS/BSA solution after each incubation. Thereafter, cells wereanalyzed by flow cytometry using the FACSort cytometer and CELLQuestsoftware (Beckton-Dickinson, Mountain View, Calif.). Three antibodieswere used for inhibition of binding in these experiments: 34-5-3(anti-I-A^(b), Pharmingen, San Diego, Calif.); MKD6 (anti-I-A^(d),Beckton-Dickinson) and 10.3.6.2 (anti-I-A^(s) (Zamvil et al., 1988)).

Example 1 Synthesis of the Peptides

[0096] The synthetic peptides of the invention of the formulas Ia, IIaand IIIa herein as well as control peptides were prepared with anautomated synthesizer (Applied Biosystem model 430A, Germany) using themanufacturer's protocols for t-butyloxycarbonyl (BOC) procedure (seeKent et al., 1984; Schnolzer et al., 1992). Briefly, in this procedure,commercially available side-chain protected amino acids were used, theamino acids being added at each step with at least 99% efficiency. Theprotecting groups were removed from the peptides and were cleared fromthe resin with anhydrous HF. Subsequently, the peptides were purified byextraction with ethyl acetate or isopropyl acetate and by HPLC. Thepurity of the peptides Ia, IIa and IIIa so obtained was then verified byHPLC and amino acid analysis.

[0097] For the preparation of peptides IVa and Va herein and analogs ofthe peptides Ia to Va of the invention, the same procedure as notedabove may be used.

[0098] The peptides Ia, IIa and IIIa were then analyzed for theirbiological activity and other characteristics as set forth in Examples2-14 below. It is to be understood that the other peptides not so-testedmay be subjected to the same analysis.

Example 2 Detection of Anti-DNA Antibodies in the Sera of Mice Immunizedwith Peptides Ia and IIIa

[0099] SJL/J and BALB/c female mice (6-8 week old) were immunized with20 μg of peptide Ia or IIIa of the invention, or with a control peptidedesignated p278 (the peptide designated Pep 278h described in publishedPCT International Application WO 94/03208) or with mAb 5G12 emulsifiedin complete Freund's adjuvant (CFA) in the foot pads. Three weeks laterthe mice received a booster injection with the same amount of peptide ormAb, in PBS. Thereafter, blood was drawn every two weeks. A fifth groupincluded non-immunized mice.

[0100]FIG. 1 depicts the anti-DNA antibodies in the sera of mice threemonths after the booster injection, and is very similar to the amount ofthe autoantibodies produced in later periods.

[0101] As shown in FIG. 1A, SJL/J mice that were immunized with thepeptide IIIa (open circles) show a high level of anti-DNA antibodies,that is higher than that of mice immunized with the whole antibody 5G12(open boxes). Low levels of anti-DNA antibodies were observed in thesera of SJL/J mice immunized with either the peptide Ia (open diamonds),control peptide p278 (open triangles) or normal non-immunized mice(crossed square).

[0102] As shown in FIG. 1B, BALB/c mice that were immunized either withthe whole antibody 5G12 (open boxes) or the peptide Ia (open diamonds)show presence of anti-DNA antibodies in the sera. However, sera ofBALB/c mice immunized with either the peptide IIIa (open circles), p278(open triangles) or normal non-immunized mice (crossed square) did notshow presence of anti-DNA antibodies.

[0103] ELISA was utilized to test the presence of the anti-DNAantibodies in the sera of the mice, as follows: Plates (Nunc) werecoated for 90 min with 10 μg/ml of methylated BSA. Thereafter the plateswere washed (all the washes were 3 times with PBS/0.05% Tween 20(Sigma)) and incubated for an additional 90 min with 10 μg/ml ofsingle-stranded DNA (calf thymus DNA (Sigma) that was heated for 15 minat 90° C. and fast-cooled). The plates were washed and blocked overnightwith 1% ovalbumin in PBS (Sigma). Thereafter, the plates were washed andincubated with the sera of the mice diluted in the blocking reagent,followed by wash and incubation with 1:500 dilution of goat anti-mouseIgG (Fc receptor specific) polyclonal antibody conjugated to peroxidase.The plates were then washed and developed using ABTS substrate (Sigma),and the color was read using an ELISA reader at 414 nm. Results areexpressed as mean OD of each mouse group (5 mice per group).

Example 3 Detection of Anti-nuclear Extract (NE) Antibodies in the Seraof Mice Immunized with the Peptides Ia and IIIa

[0104] Five groups of mice were immunized according to Example 2, andtheir sera were tested for the presence of anti-NE antibodies.

[0105] As shown in FIG. 2A, SJL/J mice immunized with the mAb 5G12 (opensquares) or with the peptide IIIa (open circles) produced a high levelof anti-NE antibodies, whereas mice immunized with the peptide Ia (opendiamonds) or p278 control peptide (open triangles), or normalnon-immunized mice (crossed squares), produced lower levels of anti-NEantibodies.

[0106] As shown in FIG. 2B, BALB/c mice immunized with the mAb 5G12(open squares) or with the peptide Ia (open diamonds) produced highlevels of anti-NE antibodies, whereas very low level of anti-NEantibodies was detected in the sera of BALB/c mice immunized with thepeptide IIIa (open circles). No anti-NE antibodies were detected in thegroup of mice immunized with p278 control peptide (open triangles) or innormal non-immunized mice (crossed squares).

[0107] ELISA was utilized to test the presence of the anti-NE antibodiesin the sera of the mice, as follows: Plates (Nunc) were coated with 5μg/ml of of HeLa cells NE for 90 min. Thereafter plates were washed andblocked, and ELISA was continued the next day, as described in Example 2for anti-DNA antibodies.

Example 4 Detection of Anti-RNP, Sm, Ro and La Antibodies in the Sera ofMice Immunized with the Peptides Ia and IIIa

[0108] The same sera of the mice as described in Examples 2 and 3 wereused for detection of anti-RNP, Sm, Ro and La antibodies.

[0109] As shown in FIG. 3A, SJL/J mice immunized with the peptide IIIa(lined box) produced extremely high levels of anti-Ro autoantibodies,antibodies that are typical for SLE in humans. High levels of anti-RNP,anti-Sm and anti-La antibodies were detected not only in SJL/J miceimmunized with the peptide IIIa (lined box), but also with the peptideIa (closed box), as compared to normal mice (open box) or to miceimmunized with the control peptide p278 (dotted box).

[0110] As shown in FIG. 3B, BALB/c mice immunized with the peptide Ia(closed box) or the peptide IIIa (lined box) produced very high levelsof anti-RNP antibodies. However, BALB/c mice immunized with the peptideIIIa (lined box) showed very low levels of anti-Sm, anti-La and anti-Roantibodies, as compared to BALB/c mice immunized with the peptide Ia(closed box) which produced detectable antibodies in the sera.

[0111] Plates were purchased as pre-coated plates and were blocked with1% ovalbumin in PBS for 2 hr. Thereafter the plates were washed as inExample 2 above, incubated in duplicates with 1:10 diluted sera, washedagain and ELISA was carried out as described in Example 2 above.

Example 5 Clinical Manifestations of SLE in Mice Immunized with thePeptides Ia and IIIa

[0112] BALB/c and SJL mice were immunized with mAb 5G12 or with peptidesIa and IIIa, and five months later were checked by two criteria formanifestation of SLE: white blood cell count (WBC) and proteinuria.

[0113] (i) White blood cell count (WBC): The mice were bled, their bloodwas diluted 1:10 with 1% (vol/vol) acetic acid in order to eliminate thered blood cells, and white blood cells were counted under a normal lightmicroscope.

[0114] Proteinuria: The urine of the mice was tested using combisticks(Combistix Kit, Ames) for the presence of protein. High levels ofprotein in the urine are indicative of kidney damage, a typicalmanifestation of SLE. TABLE 1 Clinical manifestations of mice immunizedwith the peptides Immu- BALB/c BALB/c SJL nization W.B.C.^(a)proteinuria^(b) SJL W.B.C.^(a) proteinuria^(b) mAb 5G12 3800 ± 400 0.975± 0.08 Nd^(c)  0.8 ± 0.07 pep Ia 3375 ± 350 0.88 ± 0.076 Nd^(c) 0.375 ±0.04 pep IIIa 3325 ± 400 0.30 ± 0.01 3300 ± 1343  0.9 ± 0.075 p278 6470± 920 0.33 ± 0.02 7150 ± 320  0.2 ± 0.25 non- 6800 ± 1200  0.1 ± 0 8100± 475  0.5 ± 0 immunized

[0115] The results for both WBC and proteinuria are shown in Table 1:Mice immunized with either the mAb 5G12 or the peptides Ia or IIIa had alower number of white blood cells in comparison to non-immunized mice orthose immunized with p278 control peptide. High levels of protein weremeasured in the urine of both BALB/c and SJL mice immunized with mAb5G12, of SJL mice immunized with the IIIa peptide and of BALB/c miceimmunized with the Ia peptide, while a smaller increase in protein levelwas detected in the urine of both mice immunized with control peptidep278, of IIIa-immunized BALB/c mice anf of Ia-immunized SJL mice.

Example 6 Specificity of Mice Response to the Peptides

[0116] As shown in previous examples, the peptides Ia and IIIa were usedfor the immunization of different mouse strains, in parallel to theirimmunization with the whole monoclonal antibody. The draining lymphnodes of the mice proliferated to the immunizing peptides to differentextents, depending on the mouse strains. Thus, BALB/c mice were found tobe high responders to peptide Ia, whereas SJL mice were found to be highresponders to peptide IIIa. Both peptides were used in attempts toinduce experimental SLE using the protocol utilized for the pathogenicautoantibodies. It was found that SJL mice that were immunized withpeptide IIIa and BALB/c mice that were immunized with peptide Iaproduced elevated levels of autoantibodies including anti-DNA (seeFIG. 1) and anti-NE antibodies (see FIG. 2). Moreover, the immunizedmice developed leukopenia and proteinuria (see Table 1) similarly tomice in which experimental SLE has been induced using the murineanti-DNA, 16/6Id+ pathogenic 5G12 mAb. Kidney analysis of thepeptide-injected mice revealed mild immune complex deposits in part ofthe mice. These results indicate that peptides Ia and IIIa are importantT cell epitopes of the whole molecule of the pathogenic autoantibody.

[0117] In order to assess the correlation between the peptides of theinvention and T cells, a T cell line specific to peptide IIIa of SJLorigin (high responders to the peptide IIIa) was established. The Tcells of the line proliferated specifically to the peptide IIIa but notto non-relevant control peptide p278, and upon stimulation with peptideIIIa, secreted the Thi-type cytokines, namely, IL-2, IFNγ and TNFα.Injection of the T cell line into syngeneic healthy mice led to theproduction of autoantibodies and development of clinical manifestationsthat are characteristic to mice with experimental SLE. These resultsconfirm the role of the CDR-based peptides of the invention inexperimental SLE and demonstrate the role of the peptide-specific Tcells in the autoimmune disease.

Example 7 Detection of Anti-DNA and Anti-NE Antibodies in the Sera ofBALB/c Mice Tolerized with the Peptide Ia and Immunized with EitherPeptide Ia or mAb 5G12

[0118] In order to further elucidate the role of the peptides in SLE,peptide Ia was utilized for the induction of tolerance in BALB/c mice.Newborn mice were injected twice (at day 1 and 3) with either peptide Iaor a control peptide. Thus, neonatal BALB/c mice, 24 hr old, wereinjected intraperitoneally (i.p.) with 100 μg of the peptide Ia or thecontrol peptide p307 (a peptide related to myasthenia gravis describedin published PCT Application No. WO 94/00148) in PBS, and received asecond injection 48 hr later with the same amount of peptide. Six toseven weeks after injection, the mice were immunized as described inExample 2 above with either the mAb 5G12 or the peptide Ia. The micewere bled two weeks after boost (and then periodically every two weeks)and the sera of the mice were tested for the presence of anti-DNA oranti-NE antibodies, as described in Examples 1 and 2 above. The assaysperformed to measure these autoantibody titers in the sera of theexperimental mice indicated that the mice that were tolerized withpeptide Ia did not produce significant titers of antibodies to eitherDNA or nuclear extract antigens, whereas mice tolerized to the controlpeptide p307 prior to their immunization with peptide Ia or the mAb 5G12produced high autoantibody titers.

[0119] As shown in FIGS. 4A-B, BALB/c mice that were either tolerizedwith the peptide Ia and then immunized with the mAb 5G12 (half-filledsquares), or tolerized with the peptide Ia and then immunized with thesame peptide Ia (filled squares) produced lower levels of anti-DNA andanti-NE antibodies in comparison with mice that were tolerized with thenon-relevant peptide p307 and then immunized with the mAb 5G12 (filledtriangles), or tolerized with peptide 307 and then immunized withpeptide Ia (filled circles).

[0120] This indicates that neonatal tolerization with the peptide Iacould lower the levels of autoantibodies in the sera of mice laterimmunized with the peptide Ia or the mAb 5G12.

Example 8 In vivo Inhibition of Lymph Node Cell (LNC) ProliferationResponses to the CDR-based Peptides Ia and IIIa

[0121] BALB/c (FIG. 5a) and SJL (FIG. 5b) mice were immunized withpeptides Ia and IIIa (20 μg/mouse in CFA i.d. in the hind footpads),respectively. The mice were also injected i.v. with 200 μg of the abovepeptides in PBS either 3 days prior to immunization (open squares), atthe immunization day (open circles) or at both dates (open triangles).Ten days later the mice were sacrificed and their lymph nodes wereremoved and tested for proliferation in the presence of differentconcentrations of the immunizing peptide. Control groups were of LNCtaken from mice that were immunized but not treated (filled squares), ortreated with control peptide, p307 (half filled squares). The culturemixtures were incubated for 96 hours in enriched RPMI medium containing1% normal mouse serum prior to addition of ³H-thymidine. Sixteen hourslater cells were harvested and radioactivity was counted. Results areexpressed as mean CPM of triplicates. SD values did not exceed 10%.

[0122] As shown in FIGS. 5a-b, both peptides Ia (5 a) and IIIa (5 b)inhibited proliferative responses of LNC of BALB/c and SJL mice,respectively, when injected to the mice either 3 days prior to, or atthe immunization day: Up to 95% of the proliferative capacity of thecells was inhibited by the peptides.The inhibition was specific sincethe proliferative responses of the LNC to Con A were not inhibited bypeptides Ia and IIIa (not shown).

Example 9 In vivo Inhibition of LNC Proliferation of Mice Immunized withmAb 5G12 and Treated with Peptides Ia and IIIa

[0123] BALB/c (FIG. 6a) and SJL (FIG. 6b) mice were immunized with mAb5G12 (20 μg/mouse in CFA i.d. in the hind footpads) and were injected(200 μg/mouse i.v. in PBS) with either peptide Ia or IIIa, respectively.Proliferation responses to mAb 5G12 were measured in LNC taken from micethat were immunized and not treated (filled squares), treatedconcomitantly with immunization with the control peptide p307 (halffilled squares) or treated with the appropriate CDR-based peptide Ia (6a) or IIIa (6 b) (open squares). Proliferation responses to theimmunodominant CDR-based peptide Ia and IIIa was also monitored in LNCtaken from non-treated mice (filled circles) or from mice treated withthe appropriate CDR-based peptide Ia or IIIa (open circles). Results areexpressed as mean CPM of triplicates. SD values did not exceed 10%.

[0124] As shown in FIGS. 6a-b, proliferative responses to mAb 5G12 ofLNC taken from mice treated with the appropriate CDR-based peptide wereinhibited comparing to the responses of non-treated mice.

Example 10 In vivo Inhibition of LNC Proliferation to the HumanMonoclonal Anti-DNA 16/6Id Antibody

[0125] BALB/c (FIG. 7a) and SJL (FIG. 7b) mice were immunized with humanmAb 16/6Id (1 μg/mouse in CFA i.d. in the hind footpads) and wereinjected (200 μg/mouse i.v. in PBS) with either peptide Ia or IIIa,respectively. Proliferation responses to mAb 16/6Id were measured in LNCtaken from immunized but not-treated mice (filled squares), from micetreated concomitantly with immunization with the control peptide p307(half filled squares) or from mice treated with the appropriateCDR-based peptide Ia or IIIa (open squares). Proliferation responseswere also shown to the immunodominant CDR-based peptide Ia or IIIa ofLNC taken from 16/6Id immunized non-treated mice (filled circles) orfrom mice treated with the appropriate CDR-based peptide Ia or IIIa(open circles). Results are expressed as mean CPM of triplicates. SDvalues did not exceed 10%.

[0126] As shown in FIGS. 7a-b, proliferative responses to mAb 16/6Id ofLNC taken from mice treated with the appropriate CDR-based peptide Ia orIIIa were inhibited comparing to the responses of immunized but nottreated mice, or mice treated with the control peptide p307.

Example 11 Binding of CDR-based Peptides Ia and IIIa to the Surface ofSplenic Antigen-presenting Cells (APC)

[0127] Splenic adherent cells (10⁶/100 μl/tube) isolated from BALB/c,SJL, C3H.SW or C57BL/6 mice were incubated for 16 hours withbiotinylated CDR-based peptide Ia or IIIa followed by incubation withPE-streptavidin for 30 min at 4° C. Thereafter the samples wereincubated with biotinylated anti-streptavidin and for an additionalperiod with PE-streptavidin, all at 4° C. for 30 min. After washing, thecells were analysed by flow cytometry using the FACSort cytometer andCELLQuest software.

[0128] The results are shown in FIG. 8: staining of cells that wereincubated with the biotinylated CDR-based peptides is marked by solidlines, and background staining with non-biotinylated peptide is markedby broken lines. Splenic antigen-presenting cells derived from alltested mouse strains (except for C57BL/6 mice that are resistant toinduction of SLE) showed significant binding of both CDR-based peptidesIa and IIIa to MHC class II products, indicating that their bindingcapacity agrees with the susceptibility of the mouse strains to SLEinduction.

[0129] Binding of the CDR-based peptides Ia and IIIa to APC wasdetermined as described in Materials and Methods herein and the resultsare shown in Table 2. Binding percentage was about 38-53% for allstrains, except for APC from C57BL/6 strain which showed only 19.3% and8.5% binding with peptides Ia and IIIa, respectively The binding wasinhibited by the relevant anti-Ia antibodies showing the specificity ofthe binding to MHC Class II products. The results are shown in Table 3:Inhibition of binding was specific and ranged from 60% to 100%. TABLE 2Binding of peptides Ia and IIIa to APC of mice Mouse strain H-2^(a)peptide % binding BALB/c d Ia 45.7 BALB/c d IIIa 41.3 SJL s Ia 42.3 SJLs IIIa 38.0 C3H.SW b Ia 42.3 C3H.SW b IIIa 52.9 C57BL/6 b Ia 19.3C57BL/6 b IIIa 8.5

[0130] TABLE 3 Inhibition of binding of peptides Ia and IIIa to APC byanti-Ia mAb % inhibition Mouse strain H-2 mAb pep Ia pep IIIa BALB/c danti I-A^(d) (MKD6) 76.7 100 BALB/c d anti I-A^(b) (34-5-3) 0 0 SJL santi I-A^(s) (10.3.6.2) 100 92.8 SJL s anti I-A^(d) (MKD6) 0 0 C3H.SW banti I-A^(b) (34-5-3) 60 84.4 C3H.SW b anti I-A^(d) (MKD6) 0 25 C57BL/6b anti I-A^(b) (34-5-3) 82 59.3 C57BL/6 b anti I-A^(d) (MKD6) 0 0

Example 12 Detection of Antibodies Against Peptides Ia, IIa and IIIa,and Anti-16/6 Id Antibodies in the Sera of SLE Patients and HealthyControls

[0131] Human SLE patients (32 patients) were bled and their sera weretested by ELISA for their ability to bind the peptides Ia, IIa and IIIa,a control peptide p195-212 (a myasthogenic peptide described in PCTpublication No. WO 94/00148) or mAb 5G12.

[0132] Detection of the antibodies was conducted on plates that werecoated with 10 μg/ml of peptides Ia, IIa, IIIa or p195-212 or mAb 5G12,in PBS for 2 hr, washed and blocked with 1% ovalbumin in PBS for anadditional 2 hr. ELISA was continued as described after blockage inExample 2 above, using goat anti-human IgG polyclonal antibodyconjugated to peroxidase.

[0133] As shown in FIG. 9, SLE patients exhibited significantly higherlevels of antibodies that bind either peptide Ia (open squares), IIa(open diamonds) or IIIa (open circles), or mAb 5G12 (open triangles), incomparison to healthy controls (peptide Ia-healthy—closed diamonds;peptide IIa-healthy—crossed circles; peptide IIIa-healthy—inverted opentriangles; 5G12-healthy—half filled squares). No binding could beobserved when either sera of patients or controls were tested on platescoated with the non-relevant peptide p195-212 (p195-212-SLE—crossedsquares; p195-212-healthy—half filled diamonds). The results indicate acorrelation between the whole antibody molecule and the CDR-basedpeptides on the level of antibody titers.

Example 13 Proliferation of PBL from SLE Patients and Healthy Controlsin the Presence of Human 16/6 Id mAb and Peptides

[0134] Peripheral blood lymphocytes (PBL) were isolated from the bloodof SLE patients or healthy controls using ficol gradient. Thereafter,the PBL were incubated in the presence of different concentrations ofthe peptides Ia, IIa or IIIa, or the human 16/6 Id mAb for 24 hr, when asample was taken for IL-2 measurement. The assay was continued for atotal of 7 days, and ³H-thymidine was added for the last 16 hr.Proliferation was detected by reading the amount of radioactivityincorporated into the DNA of the cells.

[0135] As is seen in Table 4, a lower proportion of the PBL taken fromSLE patients reacted to the peptides or to the 16/6 Id mAb, whencompared to the healthy controls.The results are expressed in percentageof responder (34% in the first line) and the actual number of patients(11 out of 32: 11/32)

[0136] Similar results were obtained when the levels of the IL-2produced by the PBL in the presence of the peptides or the 16/6 Id mAbwere tested, as shown in the next example. TABLE 4 Proliferation of PBLfrom SLE Patients and Healthy Controls in Presence of mAb 16/6 Id andPeptides Ia-IIIa SLE Healthy Patients Controls 16/6 Id 34% 11/32 72%18/25 pep Ia 21%  7/32 44% 11/25 pep IIa 9%  3/32 28%  7/25 pep IIIa 31%10/32 60% 15/25

Example 14 Production of IL-2 by PBL of SLE Patients and HealthyControls in the Presence of Human mAb 16/6 Id and Peptides

[0137] PBL were isolated from blood of SLE patients or healthy controlsusing ficol gradient, and were incubated as in Example 13. A sample of50 μl was removed 24 hr after the assay was started, and incubated inthe presence of IL-2 sensitive cells (CTLD) for 24 hr, after which³H-thymidine was added for 16 hr, and the plates were harvested andcounted on a beta counter.

[0138] As in Table 4, it can also be seen from Table 5 that a lowerproportion of the PBL taken from SLE patients reacted to the peptides orto the 16/6 Id mAb, when compared to the healthy controls, thusindicating that the response to the peptide corresponds to that of Tcells of the patient to the pathogenic human autoantibody. TABLE 5 IL-2Production by PBL of SLE Patients and Healthy Controls in Presence ofmAb 16/6 Id and Peptides Ia-IIIa SLE Healthy Patients Controls 16/6 Id31% 10/32  66% 17/25 pep Ia 16% 5/32 56% 14/25 pep IIa 9% 3/32 32%  8/25pep IIIa 16% 5/32 64% 16/25

References

[0139] 1. Axelrod, O. and Mozes, E. Immunobiology 172: 99, 1986.

[0140] 2. Conlon, P. J. J. Immunol. 134:1280, 1983.

[0141] 3. Fricke, H., Offen, D., Mendlovic, S., Shoenfeld, Y., Bakimer,R, Sperling, J. and Mozes, E. Internatl. Immunol. 2: 225, 1990.

[0142] 4. Fricke, H., Mendlovic, S., Blank, M., Shoenfeld, Y.,Ben-Bassat, M. and Mozes, E. Immunology 73: 421, 1991.

[0143] 5. Mendlovic, S., Brocke, S., Shoenfeld, Y., Ben-Bassat, M.,Meshorer, A., Bakimer, R. and Mozes, E. E. Proc. Natl. Acad. Sci. USA85: 2260, 1988.

[0144] 6. Mendlovic, S., Fricke, H., Shoenfeld, Y. and Mozes E. Eur. J.Immunol. 19: 729, 1989.

[0145] 7. Mendlovic, S., Brocke, S., Fricke, H., Shoenfeld, Y., Bakimer,R. and Mozes, E. Immunology 69: 228, 1990.

[0146] 8. Mozes, E., Dayan, M., Zisman, E., Brocke, S., Licht, A. andPecht, I. EMBO J. 8: 4049, 1989.

[0147] 9. Ruiz, P. J., Zinger, H. and Mozes, E. Immunol. Lett. 41: 79,1994.

[0148] 10. Shoenfeld, Y., Isenberg, D. A., Rauch, J., Madaio, M. P.,Stollar, B. D. and Schwartz, R. S. J. Exp. Med. 158: 718, 1983.

[0149] 11. Sthoeger, Z. M., Tartakovsky, B., Bentwich, Z. and Mozes, E.J. Clin. Immunol. 13: 127, 1993.

[0150] 12. Waisman, A., Mendlovic, S., Ruiz, P. J., Zinger, H.,Meshorer, A. and Mozes, E. Internal. Immunol. 5: 1293, 1993.

[0151] 13. Waisman, A. and Mozes, E. Eur. J. Immunol. 23: 1566, 1993.

[0152] 14. Zisman, E., Sela. M. and Mozes. E. Proc. Natl. Acad. Sci. USA88: 9738, 1991.

[0153] 15. Zamvil et al., J. Exp. Med. 167:1586, 1988.

1 10 20 amino acids amino acid single linear peptide /note= Xaa inposition 5 is Met, Ala or Val; Xaa in position 6 is Gln, Asp, Glu, orArg; Xaa in position 7 is Trp or Ala; Xaa in position 8 is Val or Ser;and Xaa in position 9 is Lys, Glu or Ala. 1 Thr Gly Tyr Tyr Xaa Xaa XaaXaa Xaa Gln Ser Pro Glu Lys Ser Le 1 5 10 15 Glu Trp Ile Gly 20 20 aminoacids amino acid single linear peptide /note= Xaa in position 9 is Thr,Val or Ala; Xaa in position 10 is Thr, Val or Ala; Xaa in position 11 isTyr or Phe; Xaa in position 12 is Asn or Asp; Xaa in position 13 is Glnor Glu; Xaa in position 14 is Lys or Glu; and Xaa in position 15 is Pheor Tyr. 2 Glu Ile Asn Pro Ser Thr Gly Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ly1 5 10 15 Ala Lys Ala Thr 20 20 amino acids amino acid single linearpeptide /note= Xaa in position 6 is Phe, Thr or Gly; Xaa in position 7is Leu, Ala or Ser; Xaa in position 8 is Trp or Ala; Xaa in position 9is Glu or Lys; Xaa in position 13 is Met or Ala; and Xaa in position 14is Asp, Lys or Ser. 3 Tyr Tyr Cys Ala Arg Xaa Xaa Xaa Xaa Pro Tyr AlaXaa Xaa Tyr Tr 1 5 10 15 Gly Gln Gly Ser 20 19 amino acids amino acidsingle linear peptide /note= Xaa in position 4 is Met or Ala; Xaa inposition 5 is Asn, Asp or Arg; Xaa in position 6 is Trp or Ala; Xaa inposition 7 is Val or Ser; Xaa in position 8 is Lys or Glu; Xaa inposition 9 is Gln or Ala; Xaa in position 13 is Lys or Glu; and Xaa inposition 14 is Ser or Ala. 4 Gly Tyr Asn Xaa Xaa Xaa Xaa Xaa Xaa Ser HisGly Xaa Xaa Leu Gl 1 5 10 15 Trp Ile Gly 18 amino acids amino acidsingle linear peptide /note= Xaa in position 6 is Ser or Phe; Xaa inposition 7 is Gly or Ala; Xaa in position 8 is Arg, Ala or Glu; Xaa inposition 11 is Asn or Asp; Xaa in position 12 is Tyr or Phe; and Xaa inposition 13 is Trp, His or Ala. 5 Tyr Tyr Cys Ala Arg Xaa Xaa Xaa TyrGly Xaa Xaa Xaa Gly Gln Gl 1 5 10 15 Thr Leu 20 amino acids amino acidsingle linear peptide 6 Thr Gly Tyr Tyr Met Gln Trp Val Lys Gln Ser ProGlu Lys Ser Le 1 5 10 15 Glu Trp Ile Gly 20 20 amino acids amino acidsingle linear peptide 7 Glu Ile Asn Pro Ser Thr Gly Gly Thr Thr Tyr AsnGln Lys Phe Ly 1 5 10 15 Ala Lys Ala Thr 20 20 amino acids amino acidsingle linear peptide 8 Tyr Tyr Cys Ala Arg Phe Leu Trp Glu Pro Tyr AlaMet Asp Tyr Tr 1 5 10 15 Gly Gln Gly Ser 20 19 amino acids amino acidsingle linear peptide 9 Gly Tyr Asn Met Asn Trp Val Lys Gln Ser His GlyLys Ser Leu Gl 1 5 10 15 Trp Ile Gly 18 amino acids amino acid singlelinear peptide 10 Tyr Tyr Cys Ala Arg Ser Gly Arg Tyr Gly Asn Tyr TrpGly Gln Gl 1 5 10 15 Thr Leu

1. A synthetic peptide selected from the group consisting of: (i) apeptide of at least 12 and at most 30 amino acid residues based on acomplementarity-determining region (CDR) of the heavy or light chain ofa pathogenic anti-DNA monoclonal antibody that induces a systemic lupuserythematosus (SLE)-like disease in mice (hereinafter CDR-basedpeptide), a salt or a chemical derivative thereof; (ii) an analog of aCDR-based peptide defined in (i), a salt or a chemical derivativethereof; (iii) a dual synthetic peptide comprising two such peptides of(i) or analogs of (ii) covalently linked to one another either directlyor through a short linking chain; (iv) a peptide polymer comprising aplurality of sequences of said peptide (i) or analog thereof (ii); and(v) a peptide polymer (iv) attached to a macromolecular carrier.
 2. Asynthetic peptide according to claim 1, capable of: (i) inhibitingspecifically the proliferative response and cytokine secretion of Tlymphocytes of mice that are high responders to SLE-inducingautoantibodies; or (ii) inhibiting development of SLE in mice that aresusceptible to SLE-induction by pathogenic autoantibodies.
 3. Asynthetic peptide according to claim 1 or 2, being selected from thegroup consisting of peptides having the sequences I to V herein,wherein: (i) the peptide of sequence I has the formula: T G Y Y X₁ X₂ X₃X₄ X₅ Q S P E K S L E W I G [I]

wherein X₁ is Met, Ala or Val; X₂ is Gln, Asp, Glu or Arg; X₃ is Trp orAla; X₄ is Val or Ser; and X₅ is Lys, Glu or Ala; (ii) the peptide ofsequence II has the formula: E I N P S T G G X₆ X₇ X₈ X₉ X₁₀ X₁₁ X₁₂ K AK [II] A T

wherein X₆ and X₇ are each Thr, Val or Ala; X₈ is Tyr or Phe; X₉ is Asnor Asp; X₁₀ is Gln or Glu; and X₁₁ is Lys or Glu, and X₁₂ is Phe or Tyr;(iii) the peptide of sequence III has the formula: Y Y C A R X₁₃ X₁₄ X₁₅X₁₆ P Y A X₁₇ X₁₈ Y W G Q G S [III]

wherein X₁₃ is Phe, Thr or Gly; X₁₄ is Leu, Ala or Ser; X₁₅ is Trp orAla; X₁₆ is Glu or Lys; X₁₇ is Met or Ala, and X₁₅ is Asp, Lys or Ser;(iv) the peptide of sequence IV has the formula: G Y N X₁₉ X₂₀ X₂₁ X₂₂X₂₃ X₂₄ S H G X₂₅ X₂₆ L E W I G [IV]

wherein X₁₉ is Met or Ala; X₂₀ is Asn, Asp or Arg; X₂₁ is Trp or Ala;X₂₂ is Val or Ser; X₂₃ is Lys or Glu; X₂₄ is Gln or Ala; X₂₅ is Lys orGlu, and X₂₆ is Ser or Ala; and (v) the peptide of sequence V has theformula: Y Y C A R X₂₇ X₂₈ X₂₉ Y G X₃₀ X₃₁ X₃₂ G Q G [V] T L

wherein X₂₇ is Ser or Phe; X₂₈ is Gly or Ala; X₂₉ is Arg, Ala or Glu;X₃₀ is Asn or Asp; X₃₁ is Tyr or Phe, and X₃₂ is Trp, His or Ala.
 4. Apeptide according to claim 3, having a sequence Ia of the formula: T G YY M Q W V K Q S P E K S L E W I G (Ia)


5. A peptide according to claim 3, having a sequence IIa of the formula:E I N P S T G G T T Y N Q K F K A K A T (IIa)


6. A peptide according to claim 3, having a sequence IIIa of theformula: Y Y C A R F L W E P Y A M D Y W G Q G S (IIIa)


7. A peptide according to claim 3, having a sequence IVa of the formula:G Y N M N W V K Q S H G K S L E W I G (IVa)


8. A peptide according to claim 3, having a sequence Va of the formula:Y Y C A R S G R Y G N Y W G Q T L (Va)


9. A dual synthetic peptide according to claim 1 or 2, in which twodifferent sequences I to V in claim 3 are covalently linked to oneanother either directly or through a short linking chain.
 10. A dualsynthetic peptide according to claim 9, in which two different sequencesof the peptides Ia to Va are linked covalently.
 11. A peptide polymeraccording to claim 1, containing a plurality of identical sequencesselected from the sequences I to V in claim
 3. 12. A pharmaceuticalcomposition for the treatment of systemic lupus erythematosus comprisingan effective amount of a synthetic peptide or peptide polymer accordingto any one of claims 1 to 11, and a pharmaceutically acceptable carrier.13. A pharmaceutical composition for the treatment of systemic lupuserythematosus comprising an effective amount of a mixture of at leasttwo different peptides in accordance with any one of the claims 3 to 10.14. A method for the treatment of systemic lupus erythematosuscomprising administering to a systemic lupus erythematosus patient aneffective amount of a peptide or peptide polymer according to any one ofclaims 1 to
 11. 15. A method of selecting peptides capable of inhibitingthe proliferative response of T lymphocytes from a SLE patient,comprising: (i) synthesizing a peptide of at least 12 and at most 30amino acid residues, having a sequence based on the CDR region of theheavy or light chain of a pathogenic anti-DNA monoclonal antibody thatinduces a SLE-like disease in mice, or an analog thereof; (ii) testingsaid peptide or analog for its ability to inhibit the proliferativeresponse of T cells from a SLE patient, or a T cell line or clone whichis specific to the 16/6 Id anti-DNA monoclonal antibody to which the Tcells are specific; and (iii) selecting and producing said peptide onlyif it is capable of inhibiting said proliferative response.